Ventilator-associated pneumonia (VAP) is a common and sometimes fatal complication in pediatric intensive care units (PICU). The aim of our study was to characterize the distribution and drug susceptibility of the pathogens isolated from the sputum of patients with VAP in the PICU of our hospital and to provide support to the administration of antibiotics early and reasonably in the clinic. Our study was conducted between January 2007 and December 2011 at the PICU of the Children's Hospital of Zhejiang University School of Medicine. The endotracheal aspirates were collected and transported to a microbiology laboratory within 15 min. The pathogens were routinely analyzed and identified with Vitek 60 and Kirby-Bauer disk diffusion methods. Among the 121 VAP patients, 127 pathogenic strains were isolated from sputum specimens. Gram-negative and gram-positive bacteria and fungi accounted for 64.57% (82/127), 29.92% (38/127) and 5.51% (7/127), respectively. Acinetobacter baumannii (25.61%), Escherichia coli (20.27%), Stenotrophomonas maltophilia (20.27%), Klebsiella pneumoniae (16.22%) and Pseudomonas aeruginosa (9.46%) were frequently identified isolates among gram-negative bacteria. Staphylococci were susceptible to vancomycin and linezolid. All fungi were sensitive to the antimicrobial agents. The gram-negative bacteria were more prevalent than gram-positive bacteria and fungi in VAP and demonstrated a higher drug resistance. It is important to administer antimicrobial agents early and reasonably for children with VAP. Knowledge of antibiotic resistance and the characteristics of drug resistance is important for VAP prophylaxis and treatment.